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Abstract

Introduction

It was recently suggested that heat shock protein (HSP)70, an intracellular protein,
is a potential mediator of inflammatory disease when it is released into the extracellular
compartment. Although elevated HSP70 levels have been identified in rheumatoid arthritis
(RA) synovial tissues and RA synovial fluid compared with patients with osteoarthritis
and healthy individuals, it remains unclear what role extracellular HSP70 plays in
the pathogenesis of RA. This study was conducted to investigate the effects of extracellular
HSP70 on the production of RA-associated cytokines in fibroblast-like synoviocytes
from patients with RA and to elucidate the mechanisms involved.

Methods

IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 levels in culture supernatants
were measured using enzyme-linked immunosorbent assays. Activation of mitogen-activated
protein kinases (MAPKs), such as extracellular signal-regulated protein kinases (ERKs),
c-Jun amino-terminal kinase (JNK) and p38 MAPK, was detected using Western blotting.
Nuclear translocation of nuclear factor-κB (NF-κB) and degradation of the inhibitory
protein IκBα were examined using immunohistochemistry and Western blotting.

Conclusion

Extracellular HSP70 has an anti-inflammatory effect on RA by downregulating production
of IL-6, IL-8 and MCP-1 in fibroblast-like synoviocytes, which is mediated through
inhibited activation of the MAPKs and NF-κB signal pathways.

Introduction

Rheumatoid arthritis (RA) is a chronic disease that is characterized by inflammation
of the synovial membrane and proliferation of the synovial lining, resulting in progressive
joint destruction [1]. Fibroblast-like synoviocytes (FLSs) play a crucial role in the joint inflammation
and destructive process [2]. RA FLSs respond to several proinflammatory cytokines, including IL-1, tumour necrosis
factor (TNF)-α, and exhibit characteristics of inflammatory cells that are critically
involved in various aspects of rheumatoid pathophysiology [2,3]. They synthesize and secrete proinflammatory cytokines such as IL-6, and chemokines
including IL-8 and monocyte chemoattractant protein (MCP)-1 [4-7], which play roles in mediating the inflammatory functions of FLSs. IL-6 is now recognized
to be a master cytokine that is involved not only in the RA cytokine cascade but also
in actions such as promotion of expansion and activation of T cells, differentiation
of B cells, regulation of acute phase protein genes, and regulation of chemokine production
[8,9]. IL-8 and MCP-1 are key mediators that are involved in the recruitment of neutrophils,
monocytes and lymphocytes, and play important roles in inflammatory cell infiltration
[8]. Evidence from animal models of arthritis and from RA patients has shown that blockade
of these cytokines or their receptors has beneficial effects both for inflammation
and joint destruction [10-12]. Therefore, inhibition of these inflammatory mediators production by FLSs might present
an effective target for RA treatment.

Heat shock proteins (HSPs) are a family of highly conserved intracellular proteins
that are found in all prokaryotes and eukaryotic cells. Although some HSPs are constitutively
expressed, upregulation of expression is caused by exposure to a variety of cellular
stressors, including heat shock, growth factors, inflammation and infection [13,14]. HSPs are typically regarded as intracellular proteins, and their primary function
appears to be that of intracellular molecular chaperones, contributing to the folding
of nascent proteins and denatured proteins, and thus preventing protein aggregation
under stressful stimuli [15,16]. The human stress-inducible form of the 70 kDa HSP (HSP70; Genbank: NM005345) is a many-faceted molecule. In addition to serving as a intracellular chaperone,
it is released from damaged cells or viable cells after stress, and has been found
in the bloodstream of both healthy individuals and those suffering from autoimmune
diseases and inflammatory conditions [17,18]. Recent findings indicating a role for extracellular HSP70 as a cytokine that induces
secondary proinflammatory cytokine (TNF-α, IL-1 and IL-6) production may provide insight
into the pathogenesis of autoimmune disease [16,19].

Elevated levels of the inducible form of HSP70 have been identified in RA synovial
tissues and RA synovial fluid relative to those in patients with osteoarthritis and
healthy individuals [20,21]. It is unknown whether an increase in extracellular HSP70 plays a biological role
in RA, but in animal models pre-immunization with proteins of the HSP70 family, such
as mycobacterial HSP70 and the glucose-regulated protein 78, protected animals from
experimentally induced arthritis. In adjuvant-induced arthritis in rats it was shown
that the protection conferred by mycobacterial HSP70 resulted from the induction of
IL-10 producing T cells that were capable of downregulating inflammation [22-24]. However, the precise mechanism of protection by HSP70 in RA remains unclear. Findings
in arthritis models raise the question of whether HSP70 could play a role in FLSs.
Analyses of FLSs from human patients with RA reveal significantly elevated extracellular
expression of HSP70 [25], which suggests that extracellular HSP70 may play an immunomodulatory role in FLSs.
However, the interactions of HSP70 with FLSs in RA have not previously been reported.
Also unknown are whether HSP70 exogenously regulates production by FLSs of RA-associated
proinflammatory mediators.

In the present study we report the first analysis of the effects of extracellular
human inducible HSP70 on TNF-α induced secretion by RA FLSs of the proinflammatory
cytokine IL-6 and the chemokines IL-8 and MCP-1, and we elucidate the underlying mechanism.
We find that human HSP70 inhibits TNF-α induced IL-6, IL-8 and MCP-1 secretion by
human RA FLSs. Furthermore, we demonstrate that human HSP70 suppresses the activation
of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signalling
pathways induced by TNF-α. These findings clearly reveal an anti-inflammatory effect
of human HSP70 on TNF-α-mediated inflammation and demonstrate its mechanism in RA
FLSs.

Materials and methods

Cell culture

FLSs were isolated from RA synovial tissues obtained at joint replacement surgery,
as previously described [26]. The diagnoses of RA conformed to the 1987 revised American Council of Rheumatology
criteria [27]. Briefly, tissue samples were minced and treated with 1 mg/ml collagenase for 1 to
2 hours at 37°C. After washing, the cells were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Rockville, MD, USA) supplemented with 10% heat-inactivated
foetal calf seum (Life Technologies), 100 IU/ml penicillin, 100 μg/ml streptomycin,
and 2 mmol/l L-glutamine in a humidified incubator with 5% carbon dixoide and 95%
air. After overnight culture nonadherent cells were removed, and adherent cells were
cultivated in Dulbecco's modified Eagle's medium plus 10% foetal calf seum. At confluence,
cells were trypsinized, split at a 1:3 ratio and re-cultured in the same medium. All
the experiments described here utilize FLSs between the fourth and ninth passage.
That the population of FLSs was homogeneous was determined using flow cytometry (<1%
CD11b, <1% phagocytic and <1% Fcγ receptor type II positive).

Cytokine quantification by ELISA

Following stimulation of human RA FLSs by TNF-α (R&D, Minneapolis, MN, USA) in the
presence or absence of recombinant human inducible HSP70 (StressGen, Victoria, British
Columbia, Canada; catalog# ESP555), supernatants were harvested. IL-6, IL-8 and MCP-1
levels were measured using a sandwich ELISA, following the manufacturer's instructions
(R&D). All data were normalized by cell number.

Preparation of cellular extracts

Following treatment with TNF-α, cells were harvested, washed twice with cold phosphate-buffered
saline (PBS), and nuclear and cytoplasm extracts were prepared in accordance with
the method proposed by Edgar and coworkers [28]. Briefly, the cell pellet was resuspended in 200 μl cold buffer A (10 mmol/l HEPES
[pH 7.9], 10 mmol/l KCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l DTT and 0.5 mmol/l
phenylmethylsulphonyl fluoride [PMSF]). The cells were allowed to swell on ice for
15 minutes, after which 25 μl of a 10% solution of NP-40 was added and the tube was
vortexed for 10 seconds. The homogenate was centrifuged for 30 seconds in a microfuge
to recover the cytoplasm extract in the supernatant. The nuclear pellet was resuspended
in 50 μl ice-cold buffer B (20 mmol/l HEPES [pH 7.9], 0.4 mol/l NaCl, 1 mmol/l EDTA,
1 mmol/l EGTA, 1 mmol/l DTT and 1 mmol/l PMSF) and the tube was vigorously rocked
at 4°C for 15 minutes on a shaking platform. The nuclear homogenate was centrifuged
for 5 minutes to recover the nuclear extract in the supernatant. The aliquots were
stored at -80°C. The protein concentrations of the fractions were determined using
a standard Bradford assay.

Statistical analysis

Data in the figures and text were expressed as means ± standard deviation. P < 0.05 was deemed to represent statistical significance, and the significance of differences
between groups was determined using two-tailed Student's t-test or Fisher's least significant difference test.

Results

It has been demonstrated that IL-6, IL-8 and MCP-1 are key proinflammatory mediators,
produced mainly by FLSs in the synovium, and play crucial roles in the pathophysiology
of RA [29]. TNF-α is a potent activator of production of these proinflammatory mediators in
FLSs [8,30]. We therefore analyzed the effects of human HSP70 on TNF-α induced secretion of IL-6,
IL-8 and MCP-1 in RA FLSs. As demonstrated in Figure 1, TNF-α stimulation (5 to 40 ng/ml) for 24 hours induced a dose-dependent increase
in IL-6, IL-8 and MCP-1 secretion by the RA FLSs. Peak levels of IL-6, IL-8 and MCP-1
production were noted with 20 to 40 ng/ml TNF-α. In contrast to TNF-α, human HSP70
(0.1 to 10 μg/ml) alone had no significant effects on secretion by RA FLSs of IL-6,
IL-8 and MCP-1 (Figure 1). However, when the FLSs were pretreated with different concentrations of human HSP70
for 1 hour, washed and then exposed to TNF-α (20 ng/ml) for 24 hours, secretion of
IL-6, IL-8 and MCP-1 was inhibited. As shown in Figure 2, levels of production of IL-6, IL-8 and MCP-1 were increased after TNF-α stimulation
as compared with untreated controls, and the TNF-α induced increases in IL-6, IL-8
and MCP-1 secretion were attenuated in cells treated with HSP70. The inhibitory effects
of HSP70 on IL-6, IL-8 and MCP-1 secretion were dose dependent, with prominent effects
occurring at 1 to 10 μg/ml HSP70. However, treatment with the control protein ovalbumin
[31] did not inhibit TNF-α induced IL-6, IL-8 and MCP-1 secretion (Figure 2). In addition, similar results were found when we conducted the same experiment without
washing the FLSs between HSP70 and TNF-α stimulation (Additional file 1).

Recent studies have shown that contamination of HSP70 with lipopolysaccharide might
be responsible for its stimulatory activation on macrophages and dendritic cells [32,33]. To test whether a contamination of our HSP70 preparation with lipopolysaccharide
might have been responsible for the observed effects of HSP70 on cytokine secretion
by FLSs, we applied a kinetic-turbidimetric method. We first observed that the recombinant
human HSP70 used in this study contained under 0.01 EU/μg protein (1 pg/μg) bacterial
endotoxin. Second, the following studies were conducted to exclude the possibility
that such minute amounts of lipopolysaccharide might affect cytokine secretion by
FLSs by using the lipopolysaccharide inhibitor polymyxin B and by boiling the HSP70.
Figure 3 shows that the effects of human HSP70 on IL-6, IL-8 and MCP-1 secretion were completely
inhibited by boiling (which denatures proteins but not lipopolysaccharide) but not
by polymyxin B, whereas the effects of lipopolysaccharide (100 ng/ml) on IL-6, IL-8
and MCP-1 secretion were inhibited by polymyxin B but not by boiling. In addition,
in contrast to the inhibitory effects of HSP70 on FLSs, lipopolysaccharide exhibited
slightly stimulatory effects on IL-6, IL-8 and MCP-1 secretion by FLSs (Figure 3). Consequently, we conclude that the effects of HSP70 on proinflammatory mediator
secretion in FLSs were not influenced by possible lipopolysaccharide contamination
in the preparation of HSP70.

Human HSP70 suppresses the phosphorylation of MAPKs induced by TNF-α in FLSs

TNF-α-induced inflammatory cytokine production by FLSs involves the activation of
three MAPKs, namely p38, ERK1/2 (p44/42) and JNK (p46/54) [34]. To understand fully the mechanism by which HSP70 inhibits TNF-α induced proinflammatory
mediator production by human RA FLSs, we first investigated the possible effects of
HSP70 on the phosphorylation of p38, ERK and JNK. After cells were stimulated by TNF-α,
phosphorylation levels of MAPKs were subsequently measured by Western blotting analysis
using three different kinds of phospho-specific antibodies. The results indicate that
the phosphorylation levels of all three MAPKs increased dramatically after 30 minutes
of treatment with TNF-α (20 ng/ml), which is consistent with previous reports [34]. However, the phosphorylation of all three MAPK was inhibited when FLSs were pretreated
with human HSP70 and then exposed to TNF-α for 30 minutes (Figure 4). The inhibitory effects occurred in a dose-independent manner; maximal inhibition
was achieved with 1 to 10 μg/ml HSP70. Moreover, HSP70 inhibition of the phosphorylation
of p38 MAPK was more significant as compared with its inhibition of JNK and ERK. Without
TNF-α stimulation, HSP70 alone did not significantly affect the phosphorylation of
the MAPKs in RA FLSs (date not shown). Thus, the inhibitory effects of human HSP70
on proinflammatory mediator secretion induced by TNF-α in FLSs could be attributed
to the suppression of MAPK pathways.

Figure 4. HSP70 suppresses TNF-α induced phosphorylation of MAPKs in human FLSs. Rheumatoid
arthritis (RA) fibroblast-like synoviocytes (FLSs) were incubated with heat shock
protein (HSP)70 at 0.1 to 10 μg/ml for 1 hour, and the FLSs were washed and exposed
to tumour necrosis factor (TNF)-α (20 ng/ml) for 30 minutes. The cell lysates were
immunoblotted with (a) anti-phospho-p38 (p-p38) and anti-total p38 (t-p38), (b) anti-phospho-ERK (p-ERK) and anti-total ERK (t-ERK), and (c) anti-phospho-JNK (p-JNK) and anti-total JNK (t-JNK). Antibodies (Abs) against t-p38,
t-ERK, or t-JNK served as controls. The levels of p38, extracellular signal-regulated
protein kinase (ERK), and c-Jun amino-terminal kinase (JNK) were estimated by densitometry.
Shown in the left panels are representative Western blots, and in the right panels
are presented the means ± standard deviation of three independent experiments. *P < 0.05 versus the TNF-α group. MAPK, mitogen-activated protein kinase.

Because activation and nuclear translocation of NF-κB is an essential step in the
regulation of production of cytokines [35], we first examined whether HSP70 could inhibit the TNF-α induced nuclear translocation
of NF-κB by immunofluorescence. We found that p65 subunit of NF-κB was distributed
in the cytoplasmic compartment in all cells before TNF-α stimulation. Treatment with
TNF-α (20 ng/ml) resulted in marked accumulation of p65 in nuclei after 30 minutes.
However, nuclear translocation of p65 induced by TNF-α was significantly inhibited
in cells pretreated with HSP70 (Figure 5). HSP70 alone, even at high concentrations (up to 10 μg/ml), could not induce NF-κB
nuclear translocation at all (date not shown).

Figure 5. HSP70 inhibits TNF-α induced nuclear translocation of NF-κB in FLSs, as detected using
immunocytochemistry. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs)
were incubated with heat shock protein (HSP)70 (1 μg/ml) for 1 hour, and the FLSs
were washed and exposed to tumour necrosis factor (TNF)-α (20 ng/ml) for 30 minutes.
The cells were fixed, permeabilized and incubated with rabbit anti-p65 antibody, followed
by Cy3-conjugated anti-rabbit immunoglobulin (red). The nuclei of the corresponding
cells were demonstrated by Hoechst 33258 staining. Total magnification for images
was 200×.

We further confirmed these results using a Western blotting approach by probing nuclear
and cytoplasmic FLS cell extracts using monoclonal antibodies specific for the p65
subunit of NF-κB. The results showed that nuclear translocation of p65 from the cytoplasm
to the nucleus occurred at 30 minutes after TNF-α stimulation (date not shown). Treatment
of FLSs with HSP70 for 1 hours alone did not affect the nuclear translocation of NF-κB
(date not shown). However, pretreatment of FLSs with human HSP70 for 1 hour, followed
by exposure to TNF-α for 30 minutes, caused a significant inhibition of NF-κB translocation
to the nucleus, and kept the p65 subunit of NF-κB in the cytoplasmic compartment (Figure
6a).

Figure 6. HSP70 inhibits translocation of NF-κB and degradation of IκBα. Heat shock protein
(HSP)70 inhibits tumour necrosis factor (TNF)-α induced nuclear translocation of nuclear
factor-κB (NF-κB) and degradation of IκBα in fibroblast-like synoviocytes (FLSs) detected
by Western blot. (a) Human rheumatoid arthritis (RA) FLSs were incubated with HSP70 at 0.1 to 10 μg/ml
for 1 hour, and the FLSs were washed and exposed to TNF-α (20 ng/ml) for 30 minutes.
Nuclear or cytoplasmic lysates were immunoblotted with anti-P65, anti-PCNA (proliferating
cellular nuclear antigen), or anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
PCNA and GAPDH served as controls for nuclear and cytoplasmic proteins. The levels
of p65, GAPDH and PCNA were estimated by densitometry. Shown in the left panels are
representative Western blots, and shown in the right panels are the means ± standard
deviation of three independent experiments. *P < 0.05 versus the TNF-α group. (b) Human RA FLSs were incubated with HSP70 at 0.1 to 10 μg/ml for 1 hour. Then, the FLSs
were washed and exposed to TNF-α (20 ng/ml) for 20 minutes. Cytoplasmic lysates were
immunoblotted with IκBα or anti-tubulin. Tubulin served as a control for cytoplasmic
protein. The levels of IκBα and tubulin were estimated by densitometry. Shown in the
left panel is a representative Western blot, and shown in the right panel are the
means ± standard deviation of three independent experiments. *P < 0.05 versus the TNF-α group.

Human HSP70 inhibits the TNF-α induced degradation of IκBα

In order to examine the roles played by IκBα in the NF-κB activation pathway by masking
the nuclear localization sequence, we investigated the degradation of IkBα by Western
blotting using antibodies against IκBα. To explore the mechanism by which HSP70 inhibits
the nuclear translocation of NF-κB, we determined whether HSP70 could inhibit the
TNF-α induced degradation of IκBα. TNF-α (20 ng/ml) markedly induced degradation of
IκBα at 20 minutes, and this degradation was significantly inhibited in cells pretreated
with human HSP70 (Figure 6b). It was therefore concluded that HSP70 can inhibit the degradation of IκBα and subsequent
nuclear translocation of NF-κB induced by TNF-α.

Discussion

It has been demonstrated that the HSP70 family of proteins can downregulate adjuvant
arthritis [24], and it is likely that the protection resulted from induction of self-HSP70 cross-reactive
T cells that are capable of downregulating inflammation [22]. However, the mechanism underlying the regulatory effect of HSP70 in RA is complex
and incompletely understood. Synovial FLSs play a vital role in both chronic inflammation
and joint destruction, principally through synthesis of proinflammatory cytokines
and chemokines [36], which play essentially pathogenetic roles in RA. In the present study we investigated
– for the first time – the effects of human HSP70 on secretion of the proinflammatory
cytokine IL-6 and chemokines IL-8 and MCP-1 by human RA FLSs. Our results clearly
demonstrated that human HSP70 suppressed TNF-α induced IL-6, IL-8 and MCP-1 production
in a dose-dependent manner in human RA FLSs. Moreover, we observed that HSP70 suppressed
the activation of the proinflammatory mediator associated NF-κB and MAPKs signalling
pathways induced by TNF-α. Based on these combined observations, we conclude that
extracellular human HSP70 has anti-inflammatory effects on RA, probably due to inhibitory
effects of HSP70 on production of proinflammatory cytokines and chemokines in FLSs.

The roles played by HSP70 in the immune response have been a focus of many recent
investigations [24,37]. HSP70 has been reported to have both proinflammatory and anti-inflammatory effects
in autoimmune diseases [16]. Mammalian and bacterial HSP70 have been described to activate antigen-presenting
cells directly, including macrophages and dendritic cells [17]. Such immune activation might contribute to breaking of tolerance to autoantigens,
leading to the induction of autoimmune disease [16]. HSP70 can also regulate autoimmunity indirectly by activating regulatory T cells
that control pathogenic T cells specific for self-antigens other than HSPs [24]. HSP70 has also been studied and identified as an immune target in RA. The HSP70
family of proteins has been implicated in the pathogenesis of experimental and human
arthritis. Immunization with mycobacterial HSP70 has been found to protect rats from
experimentally induced arthritis through induction of IL-10 producing T cells [22,23,38]. Intravenous or subcutaneous administration of the endoplasmic reticulum chaperone
BiP (a mammalian HSP70 family member) was also found to prevent induction of and to
treat ongoing collagen-induced arthritis [39]. Elevated levels of antibody to HSP70 have been reported in RA, and the level of
HSP70 has been shown to be enhanced in RA synovial fluid and synovial tissue [20,21,40].

Although studies on the role of extracellular HSP70 in human RA are incomplete, a
picture is emerging in which the expression of HSP70 or immune reactivity to HSP70
in RA appears to be associated with downregulation, rather than induction or propagation
of inflammation. It was recently shown that mycobacterial HSP70 treatment in vitro induced IL-10 production in monocytes from blood and synovial tissue from arthritis
patients [41]. Also, BiP stimulation of human peripheral blood mononuclear cells in vitro was found to trigger the production of anti-inflammatory cytokines [42]. However, to our knowledge, the effect of extracellular human HSP70 on human RA FLSs
has not previously been studied.

The present study revealed that human HSP70 inhibited the IL-6, IL-8 and MCP-1 expression
in RA FLSs induced by TNF-α stimulation (Figure 2), although HSP70 alone had no effect on FLSs (Figure 1). The findings suggest that self-HSP70 may have anti-inflammatory effects on RA,
which might partly be due to downregulation of proinflammatory mediators by FLSs.
However, HSP70 has also been shown to induce proinflammatory cytokines such as TNF-α,
IL-1 and IL-6 in monocytes and macrophages [16,43]. The explanation for these differences may lie in the different types and/or activation
status of cells used for the experiments. Interestingly, similar extracellular anti-inflammatory
functions for other self-HSPs have also been suggested. Treatment with human HSP60
of T cells in vitro was found to inhibit the production of proinflammatory cytokines TNF-α and interferon-γ,
and to trigger production of the anti-inflammatory cytokine IL-10, which is mediated
via Toll-like receptor 2 [44]. Treatment of human monocytes with human HSP27 exaggerated IL-10 production [45]. Treatment with human HSP10 in vitro inhibited lipopolysaccharide-induced activation of NF-κB; reduced secretion of lipopolysaccharide-induced
TNF-α, IL-6 and RANTES (regulated on activation, normal T cell activated and secreted);
and enhanced IL-10 production from human peripheral blood mononuclear cells [46].

These findings suggest that, rather than being proinflammatory, self-HSP reactivity
might be a physiological mechanism for regulating proinflammatory responses and inflammatory
diseases. It should therefore not be surprising if self-HSP70 were found to have an
anti-inflammatory effect in RA. It has been shown that inflammatory stress induces
HSP70 release from viable human FLSs and normal peripheral blood mononuclear cells
[21]. It is thus conceivable that, in the inflamed RA joint, inflammatory stress contributes
to the expression and release of HSP70, and the extracellular HSP70 may act as a natural
dimmer of inflammation, which might regulate both T cell and FLS mediated inflammation.

Thus far there exists no information about the signalling pathways that are involved
in HSP70-mediated inhibitory effects on inflammation in FLSs. Signalling pathways
that regulate proinflammatory mediator expression in RA FLSs include MAPKs and NF-κB.
Three MAPK families have been implicated as playing a role in RA, including ERK1/2,
JNK and the p38 MAPK [34]. Interestingly, all three of these MAPK families are activated in RA synovial tissue
and in cultured RA FLSs, and TNF-α has the potential to signal through all of them
[47]. Our study showed that treatment with HSP70 alone had no effect on activation of
the three MAPKs (date not shown). However, human HSP70 markedly inhibited TNF-α stimulated
p38, ERK and JNK phosphorylation (Figure 4). This inhibition was more obvious in TNF-α stimulated p38 phosphorylation. Collectively,
these date suggest that HSP70 may suppress proinflammatory mediator production in
RA FLSs via suppression of MAPK pathways.

Apart from the MAPKs, NF-κB is another key regulator of proinflammatory mediator expression
and plays an important role in the induction of inflammatory cytokines in primordial
mesenchymal cell lineages, including lymphocytes, macrophages and fibroblasts [35,48]. NF-κB is mainly composed of two subunits – p50 and p65 – and is retained in the
cytosol of nonstimulated cells by a noncovalent interaction with the inhibitory molecule
IκB. Upon stimulation by proinflammatory cytokines such as TNF-α and IL-1, IκB is
degradated and NF-κB is released and translocated to the nucleus to regulate inflammatory
gene expression [35,47]. NF-κB is also over-expressed in RA synovium [48,49], and activated in RA FLSs in response to TNF-α and IL-1 [26,47,50]. We found that the degradation of IκB and subsequent nuclear translocation of the
NF-κB subunit p65 induced by TNF-α were strongly inhibited by human HSP70. Accordingly,
we conclude that the attenuation by HSP70 of proinflammatory mediator production upon
exposure to TNF-α was at least partially mediated by the suppression of NF-κB pathway.
The mechanism by which HSP70 inhibits the TNF-α induced degradation of IκBα remains
unclear. We speculate that HSP70 in medium may bind its specific surface receptor
on the RA FLSs, and activate intracellular anti-inflammatory signal transduction pathways
such as JAK2-STAT3-SOCS3, which can inhibit the TNF-α induced degradation of IκB as
well as subsequent activation and nuclear translocation of NF-κB. Recently, Human
RA FLSs were shown to express high levels of the CD91 molecule [51], which is a known internalizing receptor for HSP70. Therefore, it is also possible
that HSP70 may interact with the cell surface via the CD91 receptor, leading to receptor
mediated endocytosis. Once taken up, HSP70 may function in the same way as does intracellular
HSP70, which exerts its chaperone functions and inhibits degradation of IκBα.

Conclusion

In this study we demonstrate a novel role for exogenous human HSP70 in suppressing
proinflammatory mediator production by human RA FLSs. The anti-inflammatory role played
by human HSP70 in human RA FLSs may be accounted for by its ability to downregulate
TNF-α induced activation of MAPK and NF-κB, two vital inflammatory signal pathways
in FLSs of inflammation in RA. The results of this study indicate that human HSP70,
which is upregulated and released in response to stress and inflammation, can function
as a downregulator of FLS-induced inflammation in RA.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

XZ and XX conceived of the study, participated in its design and coordination, and
helped to draft the manuscript. XL conceived of the study, participated in its design
and performed the statistical analysis. YZ carried out the sample collection and analysis
of data. BZ carried out the ELISA analysis. YS and ML conducted the Western blot analysis.
KW participated in immunocytochemical analysis. DRMcM helped to revise the manuscript.

Acknowledgements

This work was supported by grant from the National Natural Science Foundation of China
(30330280, 30671947), the National Basic Research Program of China (2007CB512007),
and the Specialized Research Fund for the Doctoral Program of Higher Education of
China (20060533009).